Background In systemic sclerosis (SSc), the damage of microvascular endothelial cells (ECs) and their phenotype alteration are considered early events responsible for myofibroblast activation and the development of fibrosis (1). Endothelin (ET)-1 and transforming growth factor (TGF)β1 induce ECs to acquire features of extracellular matrix-producing myofibroblasts through the induction of the endothelial-to-mesenchymal transition process (EndoMT) (2).

Objectives To compare the ability of ET-1 receptor antagonists (ET_A/BRAs), bosentan and macitentan, to support the in vitro endothelial damage repair and to contrast the EndoMT induced by ET-1 in cultured human microvascular ECs (HMVECs).

Methods HMVECs, at 3rd culture passage, were grown in endothelial cell medium (EGM2-MV). A part of these cultured HMVECs was untreated or treated with either ET-1 (100nM) or TGFβ1 (10ng/mL) for 6 days. Another part of cultured cells was treated for 1hr with bosentan (10μM) or macitentan (1μM) and then stimulated with ET-1 for 6 days (2,3). At the end of treatments, cultured HMVECs were scraped with a 200μL tip to create a linear injury, whose area was determined at baseline and after 24hrs of culture in EGM2-MV by ImageJ analysis software, to evaluate the in vitro endothelial damage repair. The gene expression of specific markers of the EndoMT, such as α-smooth muscle actin (α-SMA), fibroblast specific protein-1 (S100A4), fibronectin (FN), platelet endothelial cell adhesion molecule (CD31), and vascular endothelial (VE)-cadherin was evaluated by quantitative real time polymerase chain reaction (qRT-PCR). Statistical analysis was performed by Man-Whitney non-parametric U-test.

Results ET-1 treated HMVECs showed a significantly lower ability to promote the in vitro endothelial damage repair compared to untreated cells (p<0.05). A similar effect was observed in TGFβ1-treated cells (p<0.01). On the contrary, cultured HMVECs treated with ET_A/BRAs significantly improved the in vitro endothelial damage repair compared to ET-1-treated cells (p<0.05 for bosentan and macitentan). ET-1 significantly up-regulated the gene expression of α-SMA, S100A4, and FN (p<0.0001 for all proteins) and down-regulated that of CD31 and VE-cadherin compared to untreated cells (p<0.05, p<0.01) as observed by qRT-PCR.

Bosentan contrasted the up-regulation of α-SMA as well as significantly contrasted the up-regulation of S100A4 and FN induced by ET-1 (p<0.01, p<0.05 vs. ET-1 treated cells). Macitentan significantly reduced the ET-1-induced increase in α-SMA, S100A4, and FN (p<0.01; p<0.01; p<0.05), as well as it antagonized the down-regulation of CD31 and VE-cadherin compared to ET-1 treated cells (p<0.05 for both proteins), as observed by qRT-PCR.

Conclusions Both macitentan (at very low concentration) and bosentan seem to allow in vitro endothelial damage repair and to efficiently contrast the EndoMT induced by ET-1 in cultured HMVECs. Therefore, ET_A/B receptor antagonism might contrast the damage of ECs and their phenotype transition that seems to contribute the early phases of the fibrotic process in SSc.

1. Denton CP. Clin Exp Rheumatol.2015;33 (Suppl 92):S3–7.

2. Cipriani P et al. J Rheumatol.2015;42:1808–16.

3. Cutolo M et al. J Rheumatol.2015;42:456–63.

Disclosure of Interest None declared